Sumoylation: Molecular Biology and Biochemistry Chapter Abstracts
Chapter 1
Historical Introduction to SUMO Modification
Kirsten Jensen, Thomas Sternsdorf, Paul Freemont
SUMO is an ubiquitin-related protein that covalently binds to other proteins using a mechanism analogous to, but distinct from, ubiquitin. Here we give a brief historic overview of the discovery and functions of SUMO and the SUMO modifying machinery. In recent years, a plethora of substrate proteins for SUMO modification have been identified. However, the consequences of modification by SUMO seem to vary with the particular target protein. By summarizing the progress until now, we want to bring together the history of the field with some of the latest developments.
Chapter 2
The Molecular Evolution of SUMO Proteins
Christopher N. Larsen and Alwin Köhler
SUMOs are small globular eukaryotic proteins that are highly conserved from yeast to human. As members of a larger family of ubiquitin-like proteins, they provide us with an interesting study in the molecular evolution of post-translationally added protein modifiers. This chapter is a synopsis of eukaryotic SUMO sequences, analyzed from a molecular evolutionist perspective. To learn how protein modification pathways arose, and what molecular elements were evolutionarily important, we consider the conservation of SUMO proteins, their homologies and sequence domains, structural motifs, and perform sequence alignments to identify conserved features. We also present a phylogenic analysis to relate SUMOs. Single copies of SUMO proteins were found in unicellular organisms, including protists and also lower arthropods, but we find unique, diverse SUMO-2/3 proteins in higher plants and animals. A molecular phenogram tree of SUMO proteins suggests independent diversifications among the more loosely related SUMO-2/3 proteins. Finally, our analysis of amino acid substitutions imply that SUMO cannot be used reliably as a molecular clock. This study is an exhaustive review of SUMO sequences, and is the first systematic bioinformatic attempt at characterizing the molecular evolution of a family of ubiquitin-like proteins. We make several predictions that could be experimentally tested in the future.
Chapter 3
Molecular Mechanism of Sumoylation
Yuan Chen
Over the last several years, significant progress has been made in understanding the molecular mechanism of the sumoylation pathway. Similar to ubiquitination, this pathway requires the E1 and E2 enzymes, and in most cases, requires an E3 enzyme. Including SUMO-1 and a substrate protein, the modification pathway involves five proteins and requires several reactions that lead to the conjugation between SUMO and a substrate protein. During this process, various covalent and noncovalent complexes are formed transiently among these proteins. Up to now, structural and mechanistic studies have mainly focused on SUMO-1 and Ubc9. The sumoylation pathway has several advantages for structural and mechanistic studies compared to the homologous ubiquitination pathway. First, substrates can be small peptides instead of large proteins, and thus it is straightforward to obtain and quantify substrates for structural and enzyme kinetic analysis. Second, the modification product is well defined and homogeneous, in contrast to the heterogeneous poly-ubiquitin chains in ubiquitination reactions, so that quantitation of product formation is unambiguous. Studies of Ubc9 not only provided important insights into its mechanism in the sumoylation pathway, but also provided general information on the E2 mechanism in ubiquitination and other homologous pathways. Strategies for enzyme kinetic analysis of E2 in this multi-enzyme system will also be discussed. Finally, current information on the E1 and E3 enzymes in sumoylation, and the unique structure of a SUMO-1 hydrolase will be reviewed.
Chapter 4
SUMO Proteases
Felicity Watts
SUMO proteases have the ability both to process SUMO to the mature form and to deconjugate SUMO from target proteins. They comprise a family of cysteine proteases which contain at their active site the amino acids Cys, His and Asp. Eukaryotic organisms contain multiple SUMO proteases, with two in the budding yeast, Saccharomyces cerevisiae and at least six in mammalian cells. The SUMO proteases have different locations within the cell and this may account, at least in part, for their apparent differences in activities in vivo. This Chapter will focus on the relatedness of the different proteases, their biochemical activity and their biological roles.
Chapter 5
SUMO E3 Ligases
Andrea Pichler and Frauke Melchior
Covalent modification of proteins with members of the SUMO family can in principle be accomplished with just two enzymes, the E1 activating enzyme Aos1/Uba2 and the E2 conjugating enzyme Ubc9. This is due to Ubc9's ability to directly recognize and modify specific sites in SUMO target proteins. Modification of most targets is however very inefficient under those conditions. Over the course of the last two years, three distinct types of proteins have been identified that serve as SUMO E3 ligases for specific targets in vivo and/or in vitro. These are members of the Siz / PIAS family, the nuclear pore complex protein RanBP2/Nup358, and the polycomb group protein Pc2. While PIAS proteins resemble Ubiquitin RING finger E3 ligases, RanBP2 and Pc2 are unrelated in sequence. In this chapter, we will summarize current knowledge on the function of these proteins in sumoylation.
Chapter 6
Unraveling the SUMO-2/3 Conjugation and De-conjugation Pathways
Hisato Saitoh
There are three SUMO isoforms, SUMO-1, SUMO-2 and SUMO-3, in humans and mice. Although the mature forms of SUMO-2 and -3 are 95% identical to each other at the amino acid level, they are only 47% identical to SUMO-1. Like SUMO-1, it has been demonstrated that conjugation and de-conjugation of SUMO-2/3 are post-translational modifications of cellular proteins. During the conjugation process, the SUMO-2/3 polypeptide is added successively to acceptor proteins to form either multiple mono-SUMO-2/3 adducts or a polymerized SUMO-2/3 chain. Here, we review the properties of the SUMO-2/3 conjugation and de-conjugation pathways in vertebrate species with emphasis on the biochemical functions and the range of proteins to which SUMO-2/3 is conjugated. The current data support the concept of important distinctions between the SUMO-2/3 and SUMO-1 pathways and suggest that SUMO-2/3 is an active participant in the post-translational regulatory network of vital cellular systems.
Chapter 7
Sumoylation in Yeast
Yoshiko Kikuchi
The sumoylation system is conserved from yeast to human. By making use of this powerful genetic system, yeast sumoylation offers an excellent model system. In budding yeast, the genes encoding the components in the sumoylation pathway, Smt3, a SUMO-1 ortholog, E1 (Aos1+Uba2) and E2 (Ubc9) enzymes, and Ulp1 hydrolase, are all essential for cell viability and are necessary for passing through the G2/M boundary in the cell cycle. Major target proteins are three components of the septin ring that is located at the predicted site for cytokinesis. Studies of this conjugation pathway lead to a discovery of novel factors, such as SUMO-ligases (E3). Sumoylation and desumoylation are also involved in centromere cohesion, nucleocytoplasmic trafficking, and Rad6-dependent DNA repair. The SMT3 gene was originally isolated as a multi-copy suppressor of a temperature sensitive kinetochore protein, mif2 mutant, and the sumoylated form of topoisomerase II is involved in the centromere cohesion. PCNA is sumoylated in the S-phase, and the same acceptor site lysine residue for sumoylation is ubiquitinated by the Rad6-dependent DNA repair system upon DNA damage. In fission yeast, the sumoylation system with Pmt3 is not essential for cell viability, but it is needed for various important nuclear events.
Chapter 8
PML Nuclear Bodies and Sumoylation
Valérie Lallemand-Breitenbach and Hugues de Thé
PML nuclear bodies (PML NBs) are organized protein complexes associated with the nuclear matrix. PML recruits partners with various functions and constitutes the scaffold component of NBs. Although several reports have implicated PML in apoptosis and senescence, the exact role of PML NBs is still unknown. PML is sumoylated on three target lysines, which is a specific feature of this protein. Whereas one target site (K490) is located in the nuclear localization signal and could be implicated in nuclear import, another sumoylated lysine (K160) is absolutely required for the recruitment of NB-associated proteins. Moreover, PML can be modified both by SUMO-1 and SUMO-2/3, another level of complexity and regulation. In addition to PML, many of the NB-associated proteins and several viral proteins that target and alter PML NBs are also sumoylated. Among NB-associated proteins, several are implicated in transcription repression or activation, like the transcriptional modulator Daxx or the transcriptional activator p53, which are sequestrated and up-regulated by their recruitment onto NBs respectively. Some components of the reparation machinery are also tightly associated with PML NBs. PML sumoylation and subsequent recruitment of NB-associated proteins are modulated through the cell cycle or following stress stimulations. We can propose a general role of PML NBs either as privileged sites of activity, or as storage depots of proteins regulated by sumoylation, or finally as nuclear proteolysis sites.
Chapter 9
SUMO Modification and Nucleocytoplasmic Transport
Michael J. Matunis
Since first being discovered as a covalent modification associated with the Ran GTPase activating protein RanGAP1, the small ubiquitin-related modifier (SUMO) has been implicated as a regulator of nucleocytoplasmic transport. In keeping with this early implication, more recent studies have revealed intriguing relationships between enzymes of the SUMO modification pathway and nuclear pore complexes (NPCs), the protein structures that mediate translocation of macromolecules across the nuclear envelope. Other studies have revealed both direct and indirect effects of SUMO modification on the nucleocytoplasmic trafficking of specific cellular proteins. Despite all of the accumulated data, however, no unified model for how SUMO modification regulates nucleocytoplasmic transport has yet emerged. Rather, it appears that SUMO modification may control nucleocytoplasmic transport by a variety of mechanisms. This review summarizes the current state of knowledge about SUMO modification and its relationships to the transport of macromolecules between the nucleus and the cytoplasm. Models for how SUMO modification might be linked to regulated mRNA and protein transport are discussed.
Chapter 10
Control of Gene Expression by SUMO
David Girdwood, Michael H. Tatham and Ronald T. Hay
The small ubiquitin-like modifier (SUMO) is covalently linked to lysine residues in substrate proteins and alters the properties of the proteins to which it is conjugated. In this review we explore the consequences of SUMO conjugation in gene expression. Conjugation of SUMO to components of the transcriptional machinery does not have unique effects and it can both repress and acivate transcription. However SUMO modification of transcription factors most frequently causes repression and different mechanisms, ranging from retention in nuclear bodies to recruitment of histone deacetylases, are thought to be responsible for SUMO mediated repression.
Chapter 11
Viruses and Sumoylation
Germán Rosas-Acosta and Van G. Wilson
Viral proteins were among the first discovered substrates for sumoylation. There are currently six known sumoylated viral proteins distributed among three DNA viral families: Adenoviridae, Papillomaviridae, and Herpesviridae. All of these six viral proteins are early gene products with important regulatory roles in viral transcription or replication. Additionally, three viral proteins from two families of RNA viruses, Bunyaviridae and Retroviridae, interact with SUMO and/or Ubc9 though these viral proteins are not themselves sumoylated. There are also three viral families that encode proteases that are related to the SUMO proteases (SENPs): Adenoviridae, Asfarviridae, and Poxviridae. It is clear from the available data that viral interaction with the host sumoylation system is widespread. Typically, viruses utilize SUMO modification to regulate biological function of selected viral proteins, and the known examples will be discussed. Alternatively, it is also highly likely that viruses modulate host sumoylation to promote a cellular environment favorable for viral reproduction. This chapter will focus on the known and potential interplay between viruses and the host sumoylation system.
Chapter 12
Sumoylation of Tumor Suppressor p53 and of its Ubiquitin Ligase, Mdm2
Hideyo Yasuda
Tumor suppressor protein p53 has a critical role in carcinogenesis through its functions of regulating cell proliferation, DNA repair, and apoptosis. In response to genotoxic stress, the amount of p53 and its transactivation activity are increased as a result of its post-translational modifications including phosphorylation and acetylation. The stability of p53 is regulated by the oncoprotein Mdm2. The Mdm2 has been shown to be a ubiquitin ligase toward p53. When DNA in a cell is damaged by genotoxic stress, p53 is phosphorylated at its amino-terminus by ATM kinase and/or chk2. Mdm2 cannot bind and ubiquitinylate phosphorylated p53, and so p53 becomes stable. Both p53 and Mdm2 are modified by SUMO. PIAS1 and PIASxα function as SUMO-E3 ligases for p53, and PIAS1 and PIASxβ function for Mdm2. Site-directed mutagenesis revealed that the sumoylation site of p53 is K386. Sumoylation appears to suppress the transactivation activity of p53, because the sumoylation site mutant, K386R, has much higher activity than wild-type does. In the case of Mdm2, its sumoylation site is thought to be K182, which is a residue in the nuclear localization signal (NLS). An un-sumoylated mutant, K182R, localizes in cytoplasm, while wild-type Mdm2 is nuclear. When the SV40-NLS was fused to the N-terminal part of un-sumoylated mutant, K182R, this fusion protein localized in nucleus without sumoylation. Furthermore, Mdm2 also uses RanBP2 at nuclear pore as SUMO-E3 ligase. These data suggest that sumoylation of Mdm2 is necessary for Mdm2 to be imported into nucleus.
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